Campylobacter jejuni is a major enteric pathogen and is responsible for a large number of gastroenteritis worldwide. Clinical treatment of campylobacteriosis requires the use of fluoroquinolone or macrolide antibiotics, but antibiotic-resistant Campylobacter is increasingly prevalent, compromising the effectiveness of antibiotic therapy. Novel strategies are urgently needed to control infections caused by antibiotic-resistant Campylobacter. Among the various mechanisms utilized by Campylobacter for antibiotic resistance, the CmeABC multidrug efflux pump is a critical and predominant player in the resistance to a wide range of antimicrobials and toxic compounds. This efflux system also plays an important role in facilitating the emergence of fluoroquinolone-resistant mutants from susceptible Campylobacter under antibiotic selection. To develop novel measures to combat antibiotic-resistant Campylobacter, we have initiated studies to explore the feasibility of using antisense Peptide Nucleic Acid (PNA) to target CmeABC. Our preliminary result suggests that the antisense PNA approach has a strong potential to inhibit the expression of cmeABC and sensitize Campylobacter to antibiotics. In this application, we will further develop and evaluate the antisense PNA approach as an adjunctive therapy for antibiotic-resistant Campylobacter. The Specific aim for the R21 phase is to develop and evaluate anti-CmeABC PNAs that effectively potentiate antibiotics against antimicrobial-resistant Campylobacter and the specific aims for the R33 phase are to assess the feasibility of using anti-CmeABC PNAs as an adjunctive therapy to combat antibiotic-resistant Campylobacter in an animal model, determine the efficiency of PNAs in preventing the emergence of fluoroquinolone-resistant mutants from susceptible Campylobacter, and improve the permeability and stability of PNAs by modifying cell-penetrating peptides. The proposed work takes advantage of a novel target (CmeABC) and a novel technology (antisense PNA) and utilizes both in vitro and in vivo methodologies. Once completed, the project will have developed an effective mean to extend the utility of existing antibiotics against drug-resistant Campylobacter. Furthermore, the technical platform established in this project can be potentially adapted for other antibiotic-resistant pathogens. Thus the work will not only significantly advance the discovery of novel adjunctive therapies for antibiotic-resistant Campylobacter, but also potentially benefit the development of therapy for other pathogens.